Monoclonal antibody against necrosis marker prdx4 and use thereof

ABSTRACT

[PROBLEM] To provide a monoclonal antibody against a biomarker which shows high specificity and can be effectively used in detection and diagnosis of various lesions relevant to various kinds of carcinomas and foci of necrosis, and so forth. 
     [MEANS] A monoclonal antibody against a necrosis marker consisting the following amino acid sequence: (1) the amino acid sequence of SEQ ID NO: 1, or (2) an amino acid sequence having substitution, deletion and/or insertion of one or several amino acid residues in the amino acid sequence of (1) or sharing a homology of 90% or more with the amino acid sequence of (1), and showing the same function, activity or property as that of the amino acid sequence of (1) as a protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is Divisional of U.S. application Ser. No. 13/583,524,filed Sep. 7, 2012 (now U.S. Pat. No. 9,164,096); which is a 371National Stage of International Application No. PCT/JP2011/055356 filedMar. 8, 2011, claiming priority based on Japanese Patent Application No.2010-050106 filed Mar. 8, 2010; the contents of all of which areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a novel necrosis marker, a method fordetecting a focus of necrosis using amount of the necrosis marker, a kitfor detecting a focus of necrosis containing a compound thatspecifically reacts with the necrosis marker, a composition fordiagnostic imaging or conjugate for a therapeutic treatment comprising acompound that specifically reacts with the necrosis marker and alabeling compound or a compound effective for a therapeutic treatment, akit for diagnostic imaging or pharmaceutical composition containing theconjugate, a method for identifying a necrosis marker, which comprisesdetecting necrosis relevant to a disease, and so forth.

BACKGROUND ART

Foci of necrosis are generally originate in cell death induced in astate that supply of nutrition and oxygen to cells of various tissues isreduced due to physical injury or obstruction, loss or reduction ofblood supply. For example, in the case of solid carcinomas, when acarcinoma grows, supply of nutrition no longer spread over the wholecarcinoma tissue, and as a result, a focus of necrosis is generated.Further, myocardial infarction and cerebral infarction are alwaysaccompanied by necrosis. Furthermore, postnecrotic cirrhosis, andnecrotizing pancreatitis, in which oligotrophy and hypoxia are inducedby mal blood flow, as well as necrotizing fasciitis, which isinflammation to be aggravated in subcutaneous tissues to the fascia, andso forth are also pathological conditions accompanied by foci ofnecrosis. In addition, arteriosclerotic gangrene, diabetic gangrene, andobstructive gangrene are also diseases relating to foci of necrosis.

Since foci of necrosis badly influence on surrounding tissues and cells,if it becomes possible to detect or diagnose condition or lesion of anecrosis part, or find such an aggravated lesion and perform atherapeutic treatment for that lesion as a target, it will beindustrially useful.

However, prior art techniques concerning biomarkers for detecting afocus of necrosis are limited to the three kinds of antibodies selectedby using insoluble intracellular antigens derived from a lymphoma cellline or lung cancer cell line (Patent document 1), an antibody against asoluble nuclear matrix protein (Patent document 2), an antibody or lowmolecular weight antibody specifically binding to the nucleus extractidentified as a center of necrosis of a tumor and histone H1 (Patentdocument 3), and so forth. As markers for diagnosing severity includingnecrosis of a cardiovascular diseases-related tissue, inflammatorymarkers (CRP, TNF, IL-1, IL-6 etc.) are known. BNP is also used as amyocardial stress marker.

Further, as biomarkers of carcinomas, for example, peroxiredoxin 4 isknown as a biomarker of pancreatic cancer and lung adenocarcinoma(Non-patent documents 1 and 2), and annexin A2 is known as a biomarkerof large intestine carcinoma (Patent document 4). Furthermore,development of specific monoclonal antibodies against HMGB1, which isreleased from cell nuclei of necrotic cells, for use in therapeutictreatment is also advancing.

PRIOR ART REFERENCES Patent Documents

-   Patent document 1: Japanese Patent No. 2733658-   Patent document 2: Japanese Patent No. 3190042-   Patent document 3: Japanese Patent Unexamined Publication (KOHYO)    No. 2002-519065-   Patent document 4: Japanese Patent Unexamined Publication (KOKAI)    No. 2008-14937

Non-Patent Documents

-   Non-patent document 1: Lin J F, Xu J, Tian H Y, Gao X, Chen Q X, Gu    Q, Xu G J, Song J D, Zhao F K, Int. J. Cancer, 2007 Dec. 15; 121    (12):2596-605-   Non-patent document 2: Chen G, Gharib T G, Huang C C, Thomas D G,    Shedden K A, Taylor J M, Kardia S L, Misek D E, Giordano T J,    Iannettoni M D, Orringer M B, Hanash S M, Beer D G, Clin. Cancer    Res., 2002 July; 8(7):2298-305

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

However, any biomarker showing high specificity and effectively usablein detection and diagnosis of various lesions relating to various kindsof carcinomas and foci of necrosis has not been obtained yet.

The inventors of the present invention directed their attention to thefact that a focus of necrosis was generated in a state that nutritionand oxygen supply was reduced, then succeeded in identifying, asproteins characteristic to foci of necrosis or fragments thereof,proteins and fragments thereof that are expression products of 9 kindsof genes and having functions of such versatile necrosis markers asmentioned above from human cells cultured under undernutrition, hypoxia,high density and scaffold non-dependent conditions by using a diseaseproteomics technique based on fluorescence-labeled two-dimensionaldifferential electrophoresis (2D-DIGE), mass spectrometry (MS) andN-terminal amino acid analysis, and thus accomplished the presentinvention.

Further, it was confirmed that antibodies against the aforementionedproteins could specifically recognize foci of necrosis in sections oflesion samples of breast cancer and lung cancer, and enableddistinguishable staining of them. Furthermore, it was demonstrated forthe first time by the present invention that those antibodies labeledwith an isotope could specifically recognize circumferences of a focusof necrosis in a cancer-bearing part of a nude mouse to which cancercells were transplanted, and enabled imaging of them.

Means for Solving Problem

The present invention is thus embodied as follows.

Embodiment 1

A monoclonal antibody against a necrosis marker consisting of anexpression product of a gene encoding: the amino acid sequence of SEQ IDNO: 1, or an amino acid sequence having substitution, deletion and/orinsertion of one or several amino acid residues in the amino acidsequence of SEQ ID NO: 1 or sharing a homology of 90% or more with theamino acid sequence of SEQ ID NO: 1, and showing the same function,activity or property as that of the amino acid sequence of SEQ ID NO: 1as a protein.

Embodiment 2

The monoclonal antibody according to Embodiment 1, wherein the necrosismarker consists of a partial polypeptide of a protein having the aminoacid sequence of SEQ ID NO: 1.

Embodiment 3

The monoclonal antibody according to Embodiment 1 or 2, which isproduced by a hybridoma, NITE BP-1062 (YKP4 C8505 FCS(+)).

Embodiment 4

A method for detecting a focus of necrosis, which comprises measuringamount of a necrosis marker by using the monoclonal antibody accordingto any one of Embodiments 1 to 3.

Embodiment 5

The method according to claim 4, which comprises measuring aconcentration of a necrosis marker contained in a sample which consistsof one or more kinds of necrosis markers mentioned in any one ofEmbodiments 1 to 3, and detecting the focus of necrosis on the basis ofincrease of the measured concentration compared with a normal level asan indicator.

Embodiment 6

The method according to Embodiment 4 or 5, wherein whole blood or bloodserum is used as the sample.

Embodiment 7

The method according to any one of Embodiments 4 to 6, wherein the focusof necrosis relates to a solid carcinoma, myocardial infarction,cerebral infarction, postnecrotic cirrhosis, necrotizing pancreatitis,necrotizing fasciitis, arteriosclerotic gangrene, diabetic gangrene, orobstructive gangrene.

Embodiment 8

A detection kit used for the method according to any one of Embodiments4 to 7, which comprises the monoclonal antibody according to any one ofEmbodiments 1 to 3.

Embodiment 9

A conjugate for diagnostic imaging or a therapeutic treatment, whichconsists of a compound that specifically reacts with a necrosis markerconsisting of an expression product of a gene encoding the amino acidsequence of SEQ ID NO: 1, or an amino acid sequence having substitution,deletion and/or insertion of one or several amino acid residues in theamino acid sequence of SEQ ID NO: 1 or sharing a homology of 90% or morewith the amino acid sequence of SEQ ID NO: 1, and showing the samefunction, activity or property as that of the amino acid sequence of SEQID NO: 1 as a protein, and a labeling compound or a therapeuticallyeffective compound.

Embodiment 10

A kit for use in diagnostic imaging or a pharmaceutical composition,which comprises the conjugate according to Embodiment 9 as an activecomponent/ingredient.

Effect of the Invention

By measuring amount of the necrosis marker of the present invention in asample, not only a focus of necrosis (tissue) in various solidcarcinomas, myocardial infarction or cerebral infarction, but also afocus of necrosis in postnecrotic cirrhosis, necrotizing pancreatitis,necrotising fasciitis, etc. and further, a part to be a focus ofnecrosis in arteriosclerotic gangrene, diabetic gangrene, or obstructivegangrene can be detected.

Further, it was confirmed that foci of necrosis in sections of lesionsamples of breast cancer and lung cancer could be distinguishablystained by using an antibody against a protein as the necrosis marker ofthe present invention or a fragment thereof, and thus it was concretelydemonstrated that it is useful as a necrosis marker that can detecttissues of foci of necrosis in human breast cancer and lung cancer,positively.

Furthermore, it was demonstrated that the antibody labeled with anisotope could specifically recognize circumferences of a focus ofnecrosis in a cancer-bearing part of a nude mouse to which cancer cellswere transplanted, and thereby enabled imaging of them.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows photographs of gel on which proteomics analysis of purifiedcell extracts prepared from necrosis-induced HeLa cells and usuallycultured HeLa cells was conducted by using the fluorescence-labeledtwo-dimensional differential gel electrophoresis (2D-DIGE) method in theexamples of the present invention. FIG. 1A shows the result obtained onthe day 1 after the induction of necrosis, FIG. 1B shows the resultobtained on the day 3 after the induction of necrosis, and FIG. 1C showsthe result obtained on the day 6 after the induction of necrosis, whichwere all obtained through analysis by the 2D-DIGE method. The spotsindicated with the arrows in FIG. 1C are the spots identified by themass spectrometry and the N-terminal amino acid analysis, and the spotnumbers correspond to the spot numbers (group) mentioned in Table 1.

FIG. 2 shows typical histo-immunological staining images obtained byimmunologically staining human breast cancer and human lung cancertissues containing foci of necrosis using various kinds of antibodiesagainst necrotic focus-specific markers in the examples of the presentinvention. Among the images, the images of a are the staining images ofcancerous regions of human breast cancer tissues containing foci ofnecrosis, and the images of b are the staining images of non-cancerousregions of the same. Among the images, the images of c are the stainingimages of cancerous regions of human lung cancer tissues containing fociof necrosis, and the images of d are the staining images ofnon-cancerous regions of the same. The cancer cells including foci ofnecrosis of human breast cancer and lung cancer tissues were stronglystained with each of the antibodies, and nuclei counterstained with ahematoxylin solution were observed in some places.

FIG. 3 shows a sensorgram obtained in BIACore by kinetic analysis of theinteraction of anti-PRDX4 antibody and a biotinylated peptide.

FIG. 4 shows an autoradiographic image of DTPA-IgG before and afterpurification electrophoresed by cellulose acetate electrophoresis.

FIG. 5 shows the results of measurement of reactions of In-111-labeledmonoclonal antibodies and live HeLa cells by using a gamma counter.

FIG. 6 shows X-ray photographs showing the results of imaging incancer-bearing mice using the In-111-labeled monoclonal antibody.

FIG. 7 shows the results of autoradiography and hematoxylin-eosinstaining (HE staining) conducted for adjacent samples among a pluralityof frozen samples of tumor, which were prepared for the tumor extractedafter the imaging on the day 4 after the administration of theIn-111-labeled monoclonal antibody.

FIG. 8 shows morphology of cell in a circumferential region of a focusof necrosis in which the In-111-labeled monoclonal antibodiesaccumulated.

DESCRIPTION OF EMBODIMENTS

As described in the examples of this specification, the necrosis markerof the present invention can be obtained by identification andcomparison of proteins expressed by human necrotic cells obtained byculturing appropriate human cells (for example, those of an establishedcultured cell line such as a HeLa cell strain, which is a cell strain ofcarcinoma of uterine cervix) under undernutrition, hypoxia, high densityand scaffold non-dependent conditions and proteins expressed by the samecells cultured under usual culture conditions by using a diseaseproteomics technique based on fluorescence-labeled two-dimensionaldifferential electrophoresis (2D-DIGE), mass spectrometry (MS) andN-terminal amino acid analysis, which are known to those skilled in theart.

Therefore, the present invention also provides a method for identifyinga necrosis marker for detecting necrosis relevant to a disease. Thus,this method is characterized by comparing proteins expressed by necroticcells obtained by inducing human cells by culturing underundernutrition, hypoxia, high density and scaffold non-dependentconditions and proteins expressed by the same cells cultured under usualculture conditions, and selecting a protein existing in the necroticcells at a higher concentration (highly expressed) or a fragment thereof(limited degradation product or partial protein) as the necrosis marker.

In this specification, the “undernutrition, hypoxia, high density andscaffold non-dependent conditions” means, for example, a condition thatthe cells are cultured at a cell density of about 1×10⁷ cells/ml forabout 1 to 2 weeks without changing culture medium under an anaerobiccondition in a tube with a lid in a state that the cells are suspendedin a medium contained in the tube. On the other hand, the “usual cultureconditions” mean that, for example, the cells are cultured at 37° C. anda cell density of 2×10⁴ to 1×10⁵ cells/ml in blood serum-containing DMEMmedium (Dulbecco's modified Eagle medium) under a gas phase of 5% CO₂and 95% air and saturated humidity. Further, the “protein existing at ahigher concentration (highly expressed)” means, for example, a proteincontained in a spot showing a twice or more higher fluorescenceintensity in the fluorescence-labeled two-dimensional differential gelelectrophoresis compared with that of the cells cultured under the usualculture condition, and increasing with time in the necrosis-inducedcells.

As a result of such comparison as describe above, it was found for thefirst time that such 9 kinds (groups) of proteins as gene products asshown in Table 1 mentioned in the examples, peroxiredoxin 4, ERp29,VDAC-1, annexin A2, calreticulin, GRP78, PDIA6, HSP60 and ERp57, orpartial polypeptides thereof existed in human necrotic cells at asignificantly higher concentration compared with normal human cellscultured under the usual culture conditions. Although the genes thereofare known, it has not been known so far that they are specifically andhighly expressed in a necrosis tissue.

Therefore, the aforementioned gene expression products or autoantibodiesagainst the expression products are useful as necrosis markers.

Examples of such gene expression products usable as necrosis markersinclude, for example, proteins encoded by mRNA and cDNA of the genes, ora nucleic acid molecule containing a partial nucleotide sequencethereof, the proteins encoded by the genes and partial polypeptidesthereof. For example, the proteins expressed from the aforementionedgenes are useful as “necrotic focus-specific marker proteins” that arespecifically and highly expressed in necrosis tissues.

More specifically, there can be mentioned the following amino acidsequences as amino acid sequences of the proteins encoded by theaforementioned genes:

-   (1) the amino acid sequence of SEQ ID NO: 1,-   (2) an amino acid sequence having substitution, deletion and/or    insertion of one or several amino acid residues in the amino acid    sequence of (1) or sharing a homology of 90% or more, preferably 95%    or more, more preferably 98% or more with the amino acid sequence of    (1), and showing the same function, activity or property as that of    the amino acid sequence of (1) as a protein

Furthermore, as examples of the partial polypeptides of the proteinsencoded by the genes, the partial polypeptides of peroxiredoxin 4 (SEQID NO: 1) identified in Example 2 can be mentioned.

In order to determine homology (identity) of sequences in two amino acidsequences, the sequences are preliminarily optimized for the comparison.For example, a gap is inserted into one sequence to optimize thesequence for alignment with the other sequence. Then, amino acidresidues or nucleotide residues at each position are compared. When anamino acid residue or nucleotide residue existing at a certain positionin the first sequence is the same as that existing at the correspondingposition in the second sequence, those sequences are identical for thatposition. Homology of two sequences is indicated in terms of percentageof the number of positions at which the sequences are identical to thetotal number of positions (total amino acid or nucleotide residues).

Homology of two amino acid sequences can be determined according to theaforementioned principle by using an arbitrary method known to thoseskilled in the art. For example, it can be determined by the algorithmof Karlin and Altshul (Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990and Proc. Natl. Acad. Sci. USA, 90:5873-5877, 1993). The BLAST programutilizing the above algorithm was developed by Altshul et al. (J. Mol.Biol., 215:403-410, 1990). Further, Gapped BLAST is a program that candetermine homology with higher sensitivity compared with BLAST (NucleicAcids Res., 25:3389-3402, 1997). The aforementioned programs are mainlyused in order to search databases for a sequence showing high homologyto a given sequence. These are available, for example, on the web siteon the Internet of U.S. National Center for Biotechnology Information.

Alternatively, as homology of sequences, a value determined by using theBLAST 2 Sequences software developed by Tatiana A., Tatusova et al.(FEMS Microbiol. Lett., 174:247-250, 1999) can also be used. Thissoftware is available on the web site on the Internet of U.S. NationalCenter for Biotechnology Information, or it can also be obtained. Theprograms and parameters to be used are as follows. In the case of anamino acid sequence, as parameters used in the Blastp program, Open gap:11 and extension gap: 1 penalties, gap x_dropoff: 50, expect: 10, wordsize: 3, and Filter: ON are used. Furthermore, it is also possible toretrieve a sequence showing a homology from a database by using the moresensitive FASTA software (W. R. Pearson and D. J. Lipman, Proc. Natl.Acad. Sci. USA, 85:2444-2448, 1988). All of the values of the parametersare those used as default values on the web site.

The method for detecting a focus of necrosis according to the presentinvention comprises measuring amount of the aforementioned necrosismarker of the present invention. More specifically, the methodcomprises, for example, (1) measuring concentration of one necrosismarker selected from the necrosis markers of the present invention or anarbitrary combination thereof contained in a sample, and detecting afocus of necrosis on the basis of increase of the measured concentrationcompared with a normal level as an indicator.

The sample used for the method of the present invention is notparticularly limited concerning origin, form, etc., so long as a samplederived from a living body possibly containing any of the aforementionedexpression products from a focus of necrosis is used. Preferred examplesinclude whole blood and blood serum.

Type, original tissue, etc. of a focus of necrosis detectable by themethod of the present invention are not particularly limited, andexamples include, for example, foci of necrosis relating to any one ofvarious solid carcinomas, myocardial infarction, cerebral infarction,postnecrotic cirrhosis, necrotizing pancreatitis, necrotizing fasciitis,arteriosclerotic gangrene, diabetic gangrene, or obstructive gangrene.

The measurement of expression amount of the expression product of anyone of the genes mentioned above may be quantitative, semi-quantitative,or qualitative depending on the method and principle of the measurement.Degree of the increase serving as an indicator should be significantdifference in a parameter observable with types of the sample and thenecrosis marker used in the detection method, the principle, conditions,etc. of the measurement means and method, and so forth. For example, inthe case of quantitative measurement, it may be increase of 1.5 times ormore compared with a normal level.

Amount of a protein or a partial polypeptide encoded by any of theaforementioned genes can be measured by an arbitrary method known tothose skilled in the art. For example, it can be measured byimmunological staining such as Western blotting using a suitableantibody, methods utilizing various kinds of immunological specificreactions such as EIA, amino acid sequence analysis methods for peptidessuch the method of using a gas phase sequencer based on the Edmanmethod, and further, mass spectrometry, of which typical examplesinclude MALDI-TOF/MS, ESI Q-TOF/MS, and so forth.

Among the aforementioned methods, the methods of measuring expressionamount of a protein or a partial polypeptide thereof on the basis of anantigen-antibody reaction with an antibody specific to the protein or apartial polypeptide thereof, for example, Western blotting, and enzymeimmunoassay such as EIA, are preferred.

Furthermore, there can be mentioned the method of measuring amount ofthe necrosis marker consisting of an autoantibody against the expressionproduct on the basis of an antigen-antibody reaction using the proteinas a gene expression product of any one of the aforementioned genes, ora partial polypeptide thereof as an antigen.

Therefore, the aforementioned antibody can be prepared by a suitablemethod known to those skilled in the art by using the protein, anappropriate partial polypeptide (peptide fragment) thereof, any ofvarious derivatives or complexes thereof, or the like as an antigensubstance or an immunogen. For example, such an antigen substance or animmunogen can be administered to an appropriate animal such as mouse,rat, rabbit, goat and fowl, and a polyclonal antibody can be preparedfrom antiserum of the animal. Alternatively, a monoclonal antibody canbe prepared by a known method utilizing cell fusion according to knownmonoclonal antibody preparation methods (described in “MonoclonalAntibody”, Nagamune H. and Terada H., Hirokawa Publishing Co., 1990;“Monoclonal Antibody”, James W. Goding, Third edition, Academic Press,1996, etc.).

Furthermore, there can also be mentioned various kinds of artificialantibodies having arbitrary forms known to those skilled in the art, forexample, chimeric antibodies and humanized antibodies, which can beprepared by gene engineering techniques, as well as single chainantibodies, single chain variable region antibody fragment (scFv)consisting of the heavy chain variable domain having the heavy chain CDR1-3 and light chain variable domain having light chain CDR 1-3 bondedwith a linker, low molecular weight antibodies such as Fab fragmentantibody and Fab′ fragment antibody, and so forth. These can be easilyproduced by arbitrary methods known to those skilled in the art.

Such antibodies may be labeled with various labeling substances known tothose skilled in the art, such as enzymes, radioisotopes, nanoparticles,fluorescent dyes and metal atoms.

Further, mRNA (or cDNA) of the aforementioned genes can be amplified ordetected by the methods known to those skilled in the art, for example,various quantitative PCR methods such as RT-PCR using primers or probeappropriately designed on the basis of a nucleotide sequence of thegenes, and real-time reverse transcription PCR (real-time RT-PCR),various kinds of microarray (DNA chip) techniques, and so forth.Detection and identification of a nucleic acid molecule amplified by thePCR method can be performed by an appropriate method such as a method ofdirectly determining the nucleotide sequence (sequencing method), and acombination thereof with electrophoresis.

The nucleotide sequence of the aforementioned primers or probepreferably contains nucleotides in such a number that specific bindingwith a template is enabled, for example, 15 to 40 nucleotides, morespecifically, it preferably contains about 15 to 25 nucleotides, and itis also important that the nucleotide sequence should be such anucleotide sequence that any hairpin structure is not formed in theprimers, or a sense strand and an antisense strand do not anneal to eachother. It is also possible to use, for example, commercially availablesoftware for primer design such as Oligo™ (product of NationalBioscience Inc.).

The detection kit used for the detection method of the present inventionis characterized by comprising a compound that specifically reacts withthe necrosis marker of the present invention. Preferred examples of sucha compound include the aforementioned various antibodies.

The detection kit of the present invention can have a configurationsuitable for the object of the measurement or the principle of themeasurement. The kit may comprise, as the components thereof, forexample, the aforementioned expression product, for example, an antibodyspecific to a protein or a partial polypeptide thereof, or a protein asan expression product that is an antigen for an autoantibody against theexpression product or a partial polypeptide thereof, various kinds ofsecond antibodies (labeled antibody), and a compound that specificallyreacts with the expression product of the gene such as primers foramplification of the aforementioned mRNA (cDNA) and a probe forhybridization used in DNA chips (it consists of, for example, anucleotide sequence comprising about 10 to 100 of continuous nucleotideresidues). Furthermore, according to the configuration, use thereof,etc., the kit may contain other elements or components known to thoseskilled in the art, for example, various reagents, enzymes, buffers,reaction plates (vessel), and so forth. In addition, in order to makedetection after the PCR reaction easy, it is preferred that a labelingsubstance such as arbitrary fluorescent substances known to thoseskilled in the art is bound to an end of at least one of these primers.Examples of suitable labeling substances include, for example, radiationlabeling substances such as ³²P, fluorescent substances such as cyaninesincluding Cy3 and Cy5, 6-carboxyfluorescein (FAM),4,7,2′,4′,5′,7′-hexachloro-6-carboxyfluorescein (HEX), NED (AppliedSystems Japan), and 6-carboxy-X-rhodamine (Rox), chemiluminescentsubstances, and so forth.

The materials, instruments, apparatuses, and so forth used for each ofthe aforementioned measurements are easily available by those skilled inthe art, and procedure, conditions, etc. of each measurement operationcan be suitably determined according to manuals attached to theinstruments and apparatuses, or depending on the other conditions suchas type of cells to be used.

Furthermore, a complex consisting of a compound that specifically reactswith the aforementioned necrosis marker such as the various kinds ofantibodies against the necrosis marker already mentioned above and alabeling compound or a compound effective for a therapeutic treatment isuseful, for example, for diagnostic imaging such as PET or SPECT, or asa drug for a therapeutic treatment (conjugate: complex). Examples ofradionuclide effective for nuclear medicine diagnosis include, forexample, positron emitting nuclides (Cu-64 etc.) and gamma-ray emittingnuclides (In-111 etc.). Further, examples of the labeling compoundsinclude nuclides inducing electron anti-β decay such as ¹⁵O, ¹³N, ¹¹C,and ¹⁸F. Examples of the compound effective for a therapeutic treatmentinclude arbitrary drugs known to those skilled in the art, for example,antitumor agents, and radionuclides that emit cytotoxic β-ray (Y-90etc.).

Therefore, such a conjugate can be an active component/ingredient of akit for diagnostic imaging or a pharmaceutical composition. Such apharmaceutical composition can be formulated by the known pharmaceuticalmethods. For example, it is contemplated to formulate a pharmaceuticalcomposition by combining the conjugate with a pharmacologicallyacceptable carrier or medium, specifically, by suitably combining withsterilized water, physiological saline, vegetable oil, emulsifier,suspending agent, surfactant, stabilizer, agent for sustained release,etc., and administer the composition. The pharmaceutical composition ofthe present invention may be in the form of aqueous solution, tablet,capsule, troche, buccal tablet, elixir, suspension, syrup, nasal drop,inhalation solution, or the like. Content of the compound of the presentinvention can be appropriately determined by those skilled in the artdepending on the purpose of therapeutic treatment, route ofadministration, object of therapeutic treatment, and so forth.

EXAMPLES

Hereafter, the present invention will be explained in more detail withreference to examples. However, these examples do not limit thetechnical scope of the present invention at all. Those skilled in theart can implement various variations and modifications of the presentinvention without deviating from the technical scope of the presentinvention.

Isolation of Fatal Phenotype

There are various methods for identifying a cell having a fatalphenotype. In a necrotic cell or an apoptotic cell, change of cellmorphology or cell permeability, i.e., “blebbing” of the cell membrane,is observed. Various dyes, stains, and antibodies can be used in suchmethods, and examples include propidium iodide and antibody Apo2.7,caspase dye, and so forth, but not limited to these.

Trypan Blue Staining

Trypan blue is a blue dye. Live cells having an intact membraneeliminate trypan blue from intercellular compartments, and thereforethey are not stained. Since the cell membrane of dead cells or dyingcells has permeability, cytoplasm is stained in blue.

Propidium Iodide Staining

Propidium iodide (PI) is a fluorescent DNA-intercalated molecule. Livecells having an intact membrane eliminate PI from intercellularcompartments, and therefore they remain non-fluorescent. Dead cells ordying cells are infectible and permeable to PI, and they becomefluorescent. PI staining is used for both necrotic cells and apoptoticcells.

Annexin V

Annexin V binds to a moiety of an anionic phospholipid such asphosphatidylserine. The cell membranes of live cells have the lipidbilayer structure, and phospholipids are unevenly distributed in theinner membrane and the outer membrane of the cell membrane.Phosphatidylserine usually localizes in the inner membrane. When manycells suffer from apoptosis, this lipid dislocates to the outer membraneof cells, and is exposed. By this dislocation, externally addedfluorescence-conjugated annexin V is enabled to interact with the lipid,and thereby the cells suffering from apoptosis can be labeled. Then, byseparating dead cells not labeled with the fluorescence-conjugatedannexin V, necrotic cells can be separated.

Example 1 Preparation of Necrotic Focus Cell Model System

The method of the present invention requires a cell in which necrosis isinduced as a cell model forming a focus of necrosis. Therefore, necrosiswas induced in the cells of cervical cancer cell strain, HeLa cells, toprepare a cell model forming a focus of necrosis. The HeLa cells aregenerally adhesive cells, and 2×10⁵ to 1×10⁶ cells are cultured on a10-cm² petri dish in a scaffold-dependent manner. In usual culture, theyare cultured at 37° C. and a cell density of 2×10⁴ to 1×10⁵ cells/ml in10 ml of blood serum-containing DMEM medium (Dulbecco's modified Eaglemedium) under a gas phase of 5% CO₂ and 95% air and saturated humidity.The HeLa cells have high proliferation potency, and in the highlynutritious blood serum-containing DMEM medium, they proliferate to reachconfluent in a 10-cm² petri dish within two to three days. Since theHeLa cells are cells of a cancer cell line, even if the cells reachconfluent in a petri dish, they do not suffer from contact inhibition,and can proliferate. However, if they are excessively proliferated, theypromptly undergo cell death. In the present invention, by culturing theHeLa cells at a high density of 1×10⁷ cells/ml for about 1 to 2 weekswithout changing culture medium under an anaerobic condition in a tubewith a lid in a state that the cells are suspended in the mediumcontained in the tube without scaffold, undernutrition, hypoxia, highdensity and scaffold non-dependent conditions characteristic to theformation of a focus of necrosis were reproduced.

1.1 Induction of Necrosis in HeLa Cells

The HeLa cells were cultured in a usual manner in the DMEM medium (GibcoBRL) containing 100 units/ml of penicillin and streptomycin (Gibco BRL)and 10% FBS (JRH) contained in a 10-cm² petri dish. The proliferatedHeLa cell were removed from the 10-cm² petri dish with trypsin-EDTA(Gibco BRL), and washed twice with ice-cooled PBS(−), and 1×10⁷ cellswere transferred to a 1.5-ml tube, and cultured for 15 days in 1 ml ofthe aforementioned DMEM medium or the DMEM medium not containing 10%FBS. A pH indicator was added to the medium to confirm decrease ofoxygen concentration induced by oxygen consumption due to cellularrespiration from the day 3, but the culture was continued with the lid.

1.2 Isolation of Fatal Phenotype

1.2.1 Trypan Blue Staining

The necrosis-induced HeLa cells were collected by centrifugation, andwashed twice with ice-cooled PBS(−). The collected HeLa cells weresuspended in ice-cooled PBS(−) at a density of about 1×10⁶ cells/ml, a0.4% trypan blue/PBS buffer solution (Gibco BRL) in a 1/10 volume wasadded to the suspension and mixed, and the mixture was incubated at roomtemperature for 3 to 5 minutes, and then observed under a microscope.Colorless live cells as well as dead cells and dying cells stained inblue were counted.

Among the HeLa cells in which necrosis was induced by the aforementionedmethod, 13.9% of the cells in the serum-free DMEM medium and 20.6% ofthe cells in the blood serum-containing DMEM medium after the day 1 ofthe culture, 79.5% of the cells in the serum-free DMEM medium and 68.5%of the cells in the blood serum-containing DMEM medium after the day 2of the culture, and 91.5% of the cells in the serum-free DMEM medium and81.2% of the cells in the blood serum-containing DMEM medium after theday 3 of the culture were stained in blue, and thus confirmed to be deadcells. Thereafter, the cells were cultured for 15 days, and it wasconfirmed that about 90% of the cell population consisted of dead cells.

1.2.2 Staining of Dead Cells with Fluorescent Dye Label

As cells having a fatal phenotype, roughly speaking, those undergonenecrosis and those undergone apoptosis (programmed cell death) can besupposed. It is considered that, in a focus of necrosis, the ratio ofnecrotic cells is larger than that of apoptotic cells, and in order todistinguish the cell death, the cells were further stained with afluorescent dye. The necrosis-induced HeLa cells were collected bycentrifugation, and washed twice with ice-cooled PBS(−). To thecollected cells, 100 μl of a staining Hepes buffer solution (10 mMHepes-NaOH (pH 7.4), 140 mM NaCl, 5 mM CaCl₂) containing annexin V(Annexin-V-Fluos, final concentration: 20 μl/ml, Roche) and propidiumiodide (PI, final concentration: 1 μg/ml, Dojin Chemical Laboratory,Inc.) was added, and the mixture was incubated at room temperature for10 to 15 minutes, and observed under a fluorescence microscope(excitation: 488 nm, detection: 515 to 565 nm). The HeLa cells culturedon the 10-cm² petri dish were then cultured for 1 hour in the 10%FCS-containing DMEM medium containing 1 mM hydrogen peroxide, washedtwice with ice-cooled PBS(−), then returned to the usual 10%FCS-containing DMEM medium, and cultured for 5 hours to induceapoptosis. These HeLa cells were similarly stained with Annexin-V-Fluosand PI, and observed under a fluorescence microscope.

In the HeLa cells cultured for 6 days in the 10% FCS-containing DMEMmedium to induce necrosis, the nuclei were stained with PI. On the otherhand, in the HeLa cells in which apoptosis was induced, regions aroundthe cell membranes were strongly stained with annexin V, and a part ofthe cells were also stained with PI. The live cells prepared by usualculture were stained with neither PI nor annexin V. On the basis ofthese results, it was confirmed that the dead cells produced by theaforementioned method were necrotic cells.

Example 2 Identification of Marker Molecules Specific to Focus ofNecrosis

2.1 Preparation of Cell Extracts

The HeLa cells on the day 1 to day 6 after the induction of necrosis inthe DMEM medium containing 10% FCS and the HeLa cells cultured in theusual manner obtained in Example 1 were collected, and washed twice withice-cooling PBS(−). An ice-cooled solubilization buffer (20 mM Tris-HCl(pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% NP-40) in a volume of 500 to 1,000μl was added to the cells to suspend the cells, and the suspension wastransferred to a 1.5-ml tube. The soluble fraction of the cells wasextracted with rotating the tube at 4° C. for 30 minutes, andcentrifuged at 15,000 rpm for 15 minutes, and the supernatant wascollected as a cell extract. The collected cell extract was purified byusing 2D-Clean up kit (80-6484-51, Amersham) according to the attachedprotocol, and dissolved in an appropriate volume of a lysis buffer (30mM Tris (pH 8.5) 7 M urea, 2 M thiourea, 4% CHAPS, 5 mM magnesiumacetate), the solution was centrifuged, and the supernatant wascollected as a purified cell extract.

2.2 Quantification of Proteins

The proteins contained in the purified cell extract were quantified bythe Bradford method using Protein Assay Reagent (500-0006, Bio-Rad)according to the attached protocol. Specifically, 1 μl of 0.1 N HCl and8 μl of purified water were added to 1 μl of the purified cell extractto prepare a sample solution. As protein standard solutions, BSASolution (23209, Thermo) was diluted to concentrations of 0.25 to 1mg/ml, 1 μl of the lysis buffer and 1 μl of 0.1 N HCl were added to 1 μlof each standard solution to obtain a total volume of 10 μl. In asimilar manner, 1 μl of the lysis buffer and 1 μl of 0.1 N HCl wereadded to 8 μl of purified water to prepare a blank solution. To each ofthe sample solution, the standard solutions and the blank solution, 200μl of the Protein Assay reagent diluted 5 times was added, andsufficiently mixed, and the mixture was left at room temperature for 5minutes. Absorbance of the mixture was measured at a wavelength of 595nm within 1 hour after mixing the Protein Assay reagent, a calibrationcurve was prepared from the absorbance values of the BSA standardsolutions, and amount of proteins contained in the purified cell extractwas calculated.

2.3 Proteomics Analysis Using Fluorescence-Labeled Two-DimensionalDifferential Gel Electrophoresis (2D-DIGE) Method

The purified cell extracts prepared from the HeLa cells cultured in ausual manner and those on the days 1 to 6 after induction of necrosis inan amount of 25 μg each were separately labeled with Cy3 (CyDye DIGEFluor Cy3, GE Healthcare) and Cy5 (CyDye DIGE Fluor Cy5, GE Healthcare),and 10 mM lysine (SIGMA) was added to terminate thefluorescence-labeling reaction. Equal volumes of HeLa cell purifiedextracts labeled with Cy3 and Cy5 were mixed, a sample buffer forone-dimensional electrophoresis (8 M urea, 2% CHAPS, 0.5% IPG buffer(17-6004-40, GE), 0.002% bromothymol blue solution) was added andsufficiently mixed, the mixture was centrifuged at 15,000 rpm for 5minutes, and the supernatant was collected as a sample solution. A gelof 24 cm for the first dimensional electrophoresis (Immobiline DryStrippH 3-11NL, 17-6003-77, GE Healthcare) having an ion gradient of pH 3 to11) was swelled in the sample solution for 12 hours. The proteinscontained in the purified cell extract were separated by isoelectricfocusing by using an isoelectric focusing system for first dimensionalelectrophoresis (Ettan IPGphor 3 IEF System, GE Healthcare) withgradually elevating the voltage. Then, the intracellular solubilizedproteins were further separated according to the molecular weight by thesecond dimensional electrophoresis using a 10% polyacrylamide gel(Acrylamide: 17-1310-01, GE Healthcare). The fluorescence-labeledproteins separated by the two-dimensional electrophoresis were detectedwith a variable image analyzer (Typhoon TRIO+, GE Healthcare). The spotson the gel images were analyzed by using analysis softwere for 2D-DIGE,DeCyder (GE Healthcare), and protein expressions in the cells werecompared.

As a result of analysis of about 3,000 spots of proteins expressed inthe HeLa cells in which necrosis was induced, 0.6% of proteins of whichexpression increased compared with that observed in the cells culturedin a usual manner and 0.4% of proteins of which expression decreasedcompared with the same were observed on the day 1 after the induction ofnecrosis (FIG. 1A), and 1.1% of proteins of which expression increasedand 3.4% of proteins of which expression decreased compared with thesame were observed on the day 3 after the induction of necrosis (FIG.1B). Furthermore, as for expression of proteins of the intracellularsoluble fraction, expression of 5.1% of the proteins increased, andexpression of 2.9% of the proteins decreased compared with that observedin the cells cultured in the usual manner on the day 6 after theinduction of necrosis (FIG. 1C).

2.4 Identification of Proteins by Mass Spectrometry

The proteins of which expression amounts increased in the HeLa cells inwhich necrosis was induced were identified by MALD-TOF/MS. Specifically,the 10% polyacrylamide gel after the aforementioned two-dimensionalelectrophoresis was electrified with a constant current of 2 mA/cm² ofgel area for 90 minutes in a buffer of Tris, 6-aminocaproic acid andmethanol system to electrically blot the proteins in the gel on a PVDFmembrane (ProBlott (registered trademark), ABI). The proteins on thePVDF membrane were visualized by staining with Coomassie blue R-350(17-0518-01, GE Healthcare), and the objective spots were excised with arazor, and digested with 1 fmol of lysyl endopeptidase (125-05061, Wako)at 37° C. for 16 hours. The obtained fragmented peptides were purifiedwith a NuTip pipette tip (registered trademark, Glygen Corp.), andanalyzed by MALD-TOF/MS (Voyager-DEPRO, AB SCIEX). The obtainedfragments were compared with those in a database for protein analysis toobtain information concerning the obtained proteins.

2.5 Analysis of N-Terminal Amino Acids

N-Terminal amino acids of the proteins of which expression amountincreased in the necrosis-induced HeLa cells were decomposed one by oneby the Edman degradation to determine the primary structures.Specifically, the PVDF membranes on which the proteins obtained in thesection 2.4 mentioned above were blotted was stained with Coomassie blueR-350 (GE), and the objective spots were excised with a razor, and eachtransferred to a 1.5-ml tube. After the PVDF membrane was wetted with asmall volume of acetonitrile, a washing operation of adding 1 ml ofMilli-Q water, vigorously stirring the mixture, and then removingMilli-Q water was repeated 5 times, and the amino acid sequence wasautomatically analyzed with a full automatic protein primary structureanalyzer (PPSQ33A, Shimadzu).

As a result, among the proteins identified as markers specific to focusof necrosis, the primary sequences of N-terminal amino acids (9 aminoacids) of the peroxiredoxin 4 fragment and the ERp29 fragment wereidentified as follows.

-   (1) Peroxiredoxin 4: WETEERPRT (residues 1-9 of SEQ ID NO: 2)-   (2) ERp29: LHTKGALPL (residues 1-9 of SEQ ID NO: 4)

On the basis of the results mentioned above, among the spots showing afluorescence intensity twice or more higher than that of the HeLa cellscultured in a usual manner and increasing over time in thenecrosis-induced HeLa cells, 9 kinds (groups) of marker proteinsspecific to focus of necrosis (including limitedly degraded and partialproteins) were identified as the necrosis markers of the presentinvention as shown in Table 1 mentioned below. Some of the proteinsshown in Tables 1 and 2 have a plurality of GI (GenBank Identifier)numbers. This is because the proteins, precursors and mutants thereofshow the same results in mass spectrometry.

TABLE 1 Protein (gene) SEQ ID NO Name of identified sequence (Genbankidentifier number) 1 Peroxiredoxin 4 1 1-A) Peroxiredoxin 4: PRDX4 (gi49456297) 2 ERp29 2 2-A) Endoplasmic reticulum protein 29 isoform 1precursor (gi 5803013) 3 2-B) Chain A, Crystal structure of theprotein-disulfide isomerase related chaperone 29 (gi 192987144) 4 2-C)Endoplasmic reticulum protein 29. isoform CRA_b (gi 119618398) 3 VDAC-15 3-A) Voltage-dependent anion-selective channel protein 1 (gi 4507879)6 3-B) Porin 31HM (gi 238427) 7 3-C) Porin isoform 1 (gi 6063691) 8 3-D)Chain A, Solution structure of human Vdac-1 in Ldao Micelles (gi198443050) 9 3-E) Chain A, Structure of the human Voltage-DependentAnion Channel (gi 209447280) 4 Annexin A2 10 4-A) Annexin A2 (gi73909156) 11 4-B) Chain A, Structure of Human Annexin A2 in the presenceof calcium ions (gi 56967118) 12 4-C) Annexin A2 isoform 2 (gi 4757756)13 4-D) Chain A. Annexin A2 (gi 56966699) 14 4-E) FuII-Putative annexinA2-like protein (gi 205830271) 15 4-F) Annexin A2 isoform 1 (gi50845388) 5 Calreticulin 16 5-A) Calreticulin precursor (gi 4757900) 175-B) Calreticulin precursor variant (gi 62897681) 18 5-C) Calreticulin,isoform CRA_a (gi 119604736) 6 GRP78 19 6-A) GRP78 precursor (gi 386758)20 6-B) Heat shock 70 kDa protein 5 (g i 16507237) 21 6-C) Bip protein(gi 6470150) 22 6-D) 78 kDa glucose-regulated protein (gi 2506545)

TABLE 2 Protein (gene) SEQ ID NO Name of identified sequence (Genbankidentifier number) 7 PDIA6 23 7-A) protein disulfide isomerase-relatedprotein 5 (gi 1710248) 24 7-B) protein disulfide isomerase A6 precursor(gi 5031973) 25 7-C) unnamed protein product (gi 193785970) 26 7-D)unnamed protein product (gi 34534342) 8 Heat shock 60 kDa 27 8-A) Heatshock protein 60 (gi 77702086) protein 1 28 8-B) Chaperonin (gi31542947) 29 8-C) Mitochondrial heat shock 60 kD protein 1 variant 1 (gi189502784) 9 ERp57 30 9-A) ER-60 protease (gi 1208427) 31 9-B) Proteindisulfide isomerase (gi 860986) 32 9-C) Protein disulfide-isomerase A3precursor (gi 21361657) 33 9-D) ER-60 protein (gi 2245365) 34 9-E)Protein disulfide isomerase family A, member 3, isoform CEA_a (gi119597640) 35 9-F) Phospholipase C-alpha (gi 303618)

2.6 Identification of Proteins by Western Blotting

Among the necrotic focus-specific marker proteins shown in Table 1,which were identified by mass spectrometry and N-terminal amino acidanalysis, five kinds of them mentioned below, for which commercialantibodies were available, were confirmed by Western blotting.Specifically, non-specific adsorption on the PVDF membrane obtained inthe section 2.4 on which the proteins mentioned above were blotted wasblocked by using Block Ace (UK-B80, DS Pharma Biomedical), then primaryantibodies diluted with PBS(−) to an optimum concentration (100 to 2,000times) were added, and the reaction was allowed at room temperature for1 hour with gentle shaking. After three times of washing with PBST, abiotin-labeled anti-IgG antibody (BA-1400, VECTASTAIN) was added, andthe reaction was allowed at room temperature for 30 minutes with gentleshaking. After three times of washing with PBST, anavidin/biotin-labeled horseradish peroxidase complex solution (VEC) wasadded, and the reaction was allowed at room temperature for 30 minuteswith gentle shaking. After three times of washing with PBST, a solutionof 3,3′-diaminobenzidine (DAB, SK-4105, VEC), which is a substrate ofthe peroxidase, was added, and when a color development signal wasobserved, the PVDF membrane was transferred into Milli-Q water toterminate the color development. As negative controls, Western blottingwas performed in the same manner by using a blocking peptide of anti-Bipantibody (#1084, Cell Signaling) for the anti-Bip antibody, anti-rabbitIgG control antibody for the anti-peroxiredoxin 4, anti-calreticulin,and anti-ERp57 antibodies, and anti-rabbit IgG control antibody for theanti-ERp29 antibody.

Primary Antibodies Used

-   (1) Peroxiredoxin 4: peroxiredoxin 4 antibody [7A1], ab 16943, Abcam    (spot number 1)-   (2) ERp29: ERp29 antibody, ab40982, Abcam (spot number 2)-   (3) Calreticulin: calreticulin mouse mAb, ab54922, Abcam (spot    number 5)-   (4) Bip: Bip (C50B12) Rabbit mAb, #3177, Cell Signaling (spot number    6)-   (5) ERp57: Monoclonal anti-ERp57 (TO-2), E5031, SIGMA (spot number    9)

By Western blotting, the five spots among the nine spots identified byMS and N-terminal amino acid analysis were confirmed to be those ofperoxiredoxin 4, ERp29, calreticulin, Bip, and ERp57.

Example 3 Preparation of Monoclonal Antibodies

3.1 Sensitization of Immunized Animal and Cell Fusion

For the peroxiredoxin 4 fragment and the ERp29 fragment among theproteins identified as necrotic focus-specific markers, mouse monoclonalantibodies that could recognize each sequence were prepared on the basisof the N-terminal amino acid sequences identified in Example 2.

A peptide of 10 residues consisting of the aforementioned N-terminus 9amino acid residues of the peroxiredoxin 4 fragment or the ERp29fragment and a cysteine residue added to the C-terminus of the foregoingresidues for binding with a carrier polymer (KLH) was chemicallysynthesized in a conventional manner in an amount of about 10 mg, andpurified by HPLC, and the purified peptide was used as an antigenpeptide. The antigen peptide was crosslinked with KLH in a conventionalmanner, and mixed with an equal amount of the Freund's complete adjuvant(RM606-1, Mitsubishi Chemical Iatron), and the mixture was emulsifiedand used as an immunogen. The immunogen was administered to female miceof groups each consisting of two mice in an amount of 50 μg per mouse.As the immunization, short-term immunization consisting of 3 times ofsubcutaneous administration of the immunogen into foot pads of miceevery two days and following sensitization for ten days, and long-termimmunization consisting of 3 times of subcutaneous administration of theimmunogen into the same every one week and following sensitization for 3weeks were performed. In the case of the short-term immunization, a fewhypertrophied lymph nodes were extracted on the day 10 after the startof the immunization, and the lymphocytes contained in them were fusedwith the myeloma cells P3U1. In the case of the long-term immunization,mouse lymph nodes were extracted on the day 21 after the start of theimmunization in a manner similar to that used for the short-termimmunization, and the lymphocytes contained in them were fused with themyeloma cells P3U1.

As a result of the cell fusion of the lymphocytes prepared from each twomice subjected to the short-term immunization and the long-termimmunization with the immunogen consisting of the antigen peptide of theperoxiredoxin 4 fragment or the ERp29 fragment crosslinked with KLH andthe myeloma cells of the cell strain P3U1, colonies were formed in about800 wells for each case.

3.2 Screening of Hybridomas by ELISA

The hybridomas obtained by the cell fusion were cultured on a 96-wellplate, and selected by using a usual HAT medium. The screening wasperformed by determining whether the culture supernatant of the well inwhich colony formation was observed contained antibodies that bound withthe antigen peptide of the immunogen by ELISA. A peptide consisting ofthe N-terminal peptide of the peroxiredoxin 4 fragment+cysteine(WETEERPRTC; SEQ ID NO: 2) or the N-terminal peptide of the ERp29fragment+cysteine (LHTKGALPLC; SEQ ID NO: 4), or an N-terminal 8-residuepeptide of the peroxiredoxin 4 fragment (WETEERPR; residues 1-8 of SEQID NO: 2) or an N-terminal 8-residue peptide of the ERp29 fragment(LHTKGALP; residues 1-8 of SEQ ID NO: 4), which were newly chemicallysynthesized and purified by HPLC, was diluted with PBS(−) to aconcentration of 200 μg/ml, and the solution was added to wells of a96-well immunoplate (Maxisorp, Nunc) in a volume of 50 μl per well. Theplate was left standing at 4° C. overnight to fix the synthetic peptideto the plate, washed twice with PBS(−), blocked at room temperature for1 hour with Block Ace (DS Pharma Biomedical), and washed twice withPBS(−) containing 0.1% Triton-X 100 (PBST). The culture supernatant (100μl) of each well in which colony formation was observed was added, andthe plate was incubated at room temperature for 1 hour, and then washedtwice with PBST. After the reaction with biotin-labeled anti-mouse IgGantibody (BA-1400, VEC) was allowed at room temperature for 30 minutes,the plate was washed 3 times with PBST. Further, the reaction with anavidin/biotin-labeled peroxidase complex solution (Mouse IgG ABC Kit,VECTASTAIN) was allowed at room temperature for 30 minutes, the platewas washed 3 times with PBST, then a solution of2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, SK-4500,VEC), which is a substrate of the peroxidase, was added, and incubationwas performed at room temperature until color development was observed.Then, an absorption signal generated by each clone was evaluated bymeasuring the absorbance at a wavelength of 405 nm using a microplatereader. Wells showing a high titer were selected, and monoclonalantibody-producing hybridomas were cloned by the limiting dilutionmethod.

As a result of the primary screening of the hybridoma culturesupernatants obtained from the peroxiredoxin 4 fragment-immunized mice,antibodies that recognized the antigen peptide consisting of theN-terminal peptide of the peroxiredoxin 4 fragment (9 amino acidresidues) to which cysteine was added, and did not recognize KLH wereproduced in 2 wells in the case of the short-term immunization, and 33wells in the case of the long-term immunization. Since the identifiedN-terminus of the peroxiredoxin 4 fragment differed from the site to becleaved by usual limited degradation, antibodies that recognized thisN-terminus sequence were considered to be necrotic focus specific. Inorder to further limit the recognition site, two kinds of peptides fornegative screening of which N-terminus was masked ((Ac) WETEERPRTC (SEQID NO: 2), GAVQGWETEERPRTC (SEQ ID NO: 3)) were chemically synthesizedin a conventional manner, purified by HPLC, crosslinked with KLH, fixedon a 96-well plate and used to perform secondary screening by ELISA. Asa result, antibodies that recognized the identified N-terminus(WETEERPRTC; SEQ ID NO: 2), but did not recognize the two kinds ofpeptides for negative screening crosslinked with KLH ((Ac)WETEERPRTC(SEQ ID NO: 2)-KLH and GAVQGWETEERPRTC (SEQ ID NO: 3)-KLH) and KLH wereproduced in 1 well in the case of the short-term immunization, and 31wells in the case of the long-term immunization, among the wellsselected by the primary screening. Among these wells, antibodies thatfurther recognized N-terminal 8 amino acid residues were produced in 3wells in the case of the long-term immunization, but no well in the caseof the short-term immunization. Therefore, from the wells of thehybridomas obtained from the mice subjected to the long-termimmunization, 5 wells showing a high titer including the three wells inwhich antibodies that recognized the N-terminal 8 amino acid residueswere selected, and cloning was performed by the limiting dilution methodto obtain 5 clones of anti-peroxiredoxin 4-specific monoclonalantibody-producing hybridomas.

Further, as a result of the primary screening of the hybridomasupernatants obtained from the mice immunized with the ERp29 fragment,antibodies that recognized the antigen peptide consisting of theN-terminal peptide of the ERp29 fragment (9 amino acid residues) towhich cysteine was added, and did not recognize KLH were produced in 39wells in the case of the short-term immunization, and 21 wells in thecase of the long-term immunization. Among these wells, antibodies thatfurther recognized the N-terminal 8 amino acid residues were produced in7 wells in the case of the short-term immunization, and 5 wells in thecase of the long-term immunization. Among them, two wells showing a hightiter and derived from the short-term immunization, and three wellsshowing a high titer and derived from the long-term immunization wereselected, and cloning was performed by the limiting dilution method toobtain 5 clones of anti-ERp29 fragment-specific monoclonalantibody-producing hybridomas.

Example 4 Staining of Necrotic Focus and Circumferential Tissues ThereofUsing Monoclonal Antibodies and Commercially Available Antibodies

4.1 Histo-Immunological Staining

The antibodies that recognized the necrotic focus-specific markerproteins were evaluated by using tissue sections of cancerous regionsand non-cancerous regions of human breast cancer tissues (N=5), andcancerous regions and non-cancerous regions of human lung cancer tissues(N=5). Paraffin-embedding sections of tissue samples derived from breastcancer patients and lung cancer patients were obtained from Medical &Biological Laboratories. The paraffin embedding sections of the tissueswere deparaffinized in a conventional manner by using xylene andethanol, and the tissue sections were washed 3 times with PBS(−). Theantigen was activated with a 10 mM citrate buffer (pH 6.0), endogenousperoxidase was inactivated by leaving the sections standing in 0.3% H₂O₂for 30 minutes, and non-specific adsorption was blocked with horseserum. The antibodies against a necrotic focus marker (monoclonalantibody that recognized the N-terminal peptide of the peroxiredoxin 4fragment or the N-terminal peptide of ERp29 fragment (one clone amongeach 5 clones finally obtained in Example 3), and the five kinds ofcommercial antibodies mentioned in Example 2) were each diluted withPBS(−) to an optimum concentration (250 to 1,000 times), and reactedwith the blocked tissue section at room temperature for 1 hour, thesection was washed 3 times with PBS(−), and the reaction with abiotin-labeled anti-IgG antibody (BA-1400, VEC) was allowed at roomtemperature for 30 minutes. After the tissue section was washed 3 timeswith PBS(−), the reaction with an avidin/biotin-labeled horseradishperoxidase complex (VEC) was allowed at room temperature for 30 minutes.After the tissue section was washed 3 times with PBS(−), a solution of3,3′-diaminobenzidine (DAB, SK-4105, VEC), which is a substrate of theperoxidase, was added, and the section was incubated at room temperaturefor about 2 minutes to immunologically stain the tissue. The tissuesection that developed the color was immersed in distilled water toterminate the reaction, then counterstaining was performed with ahematoxylin solution, and the tissue was enclosed in a non-aqueousmounting medium (MGK-S, Matsunami Glass), and observed under amicroscope.

The results of the histo-immunological staining are shown in FIG. 2. Asa result of immunological staining of cancerous regions andnon-cancerous regions of human breast cancer tissues (N=5), andcancerous regions and non-cancerous regions of human lung cancer tissues(N=5) using the monoclonal antibody that recognized the N-terminalpeptide of the peroxiredoxin 4 fragment or the N-terminal peptide ofERp29 fragment, strong staining was observed in the foci of necrosis inhuman breast cancer tissues and human lung cancer tissues andcircumferential tissues thereof (FIG. 2A, a and c, and FIG. 2B, a andc), whereas staining was not observed or was weak, even if observed, inthe tissues of the non-cancerous regions of human breast cancer and lungcancer (FIG. 2A, b and d, and FIG. 2B, b and d). Further, as a result ofthe histo-immunological staining of the human tissues performed in asimilar manner by using the five kinds of commercial antibodies (primaryantibodies) mentioned in Example 2, similar results were obtained withthose antibodies, that is, strong staining was observed in the foci ofnecrosis of the human breast cancer tissues and human lung cancertissues, and circumferential tissues thereof, whereas staining was notobserved or was weak, even if observed, in the human tissues of thenon-cancerous regions (FIGS. 2C to 2G).

As a typical clone of the clones of the aforementionedanti-peroxiredoxin 4-specific monoclonal antibody-producing hybridomas,the hybridoma that produces a mouse monoclonal antibody against thehuman peroxiredoxin 4 (PRDX4) fragment as a necrosis marker (code: YKP4C8505 FCS(+)) was deposited at the Incorporated Administrative Agency,National Institute of Evaluation, Patent Microorganisms Depositary,2-5-8 Kazusakamatri, Kisarazu-shi, Chiba, 292-0818, Japan under theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure. Thedeposition was confirmed by this depositary with an accession number ofNITE BP-1062 (receiving date: Jan. 20, 2011).

Example 5 Purification of Monoclonal Antibody

The anti-human PRDX4 monoclonal antibody was purified from the culturesupernatant of the hybridoma YKP4 C8505 FCS(+) with a protein A column.Specifically, 50 ml of the culture supernatant was added to 1 ml ofHiTrapA column equilibrated with PBS(−), and after the column was washedwith 5 ml of PBS(−), eluted from the column with a 0.17 M buffer (pH2.3). One-milliliter eluted fractions were collected with monitoringabsorbance at 280 nm, and proteins in the fractions showing a highabsorbance were quantified by the Bradford method (Bio-Rad), and apurified monoclonal antibody was obtained.

Example 6 Determination of Isotype of Monoclonal Antibody

Isotype of the anti-human PRDX4 monoclonal antibody purified in Example5 was confirmed by using a mouse monoclonal antibody isotyping kit(MMT1, Serotec). The purified monoclonal antibody (150 μl) was added toa reaction tube, and reacted at room temperature for 30 secondsaccording to the attached manual. The reaction mixture was sufficientlymixed on a vortex mixer, and then an isotyping strip was put into thereaction tube to determine the isotype. As a result, the isotype of theanti-human PRDX4 monoclonal antibody was determined to be IgG1/κ.

Example 7 Evaluation of Antibody

Affinity constant and dissociation constant of the purified monoclonalantibody obtained in Example 5 were obtained by measuring the surfaceplasmon resonance phenomenon (SPR) using BIACore System (Biacore ABCorporation, currently supplied by GE Healthcare Japan, Inc.).Specifically, a biotinylated peptide used as an antigen was obtained bysynthesis, and 100 μl of a solution of the synthesized peptide obtainedby diluting the peptide at 1 mg/ml with the HB buffer was fixed onSensor Chip SA according to the instrumental manual of BIACore System. Atwo-fold serial dilution series of the purified monoclonal antibody wasprepared in a concentration range of 0.25 to 80 μg/ml, and 45 μl of eachdilution was supplied to BIACore System (flow rate: 30 μl/minute) tomeasure amount of the antibody binding to the peptide serving as theantigen according to the instrumental manual of BIACore System. As aresult, values of 1.26×10⁸ and 7.9×10⁻⁹ were obtained as the affinityconstant and dissociation constant of the anti-human PRDX4 monoclonalantibody (FIG. 3).

Example 8 Detection of Cancerous Region in Cancer-Bearing Mouse UsingRadiation-Labeled Monoclonal Antibody

-   -   1) Labeling of Anti-Human PRDX4 Monoclonal Antibody with In-111

Each IgG antibody dissolved in a 0.05 M borate buffer (pH 8.5) andN—[(R)-2-amino-3-p-isothiocyanato-phenyl]propyl]-trans-(S,S)-cyclohexane-1,2-diamine-pentaaceticacid (DTPA) dissolved in 0.05 M borate buffer (pH 8.5) were mixed at aratio of 1:2.5 on a molecular weight basis, and the reaction was allowedat 37° C. for 16 hours. Indium chloride ([In-111]Cl₃) was mixed with anequal amount of 1 M acetate buffer (pH 6.0), the mixture was leftstanding at room temperature for 5 minutes and mixed with an equalamount of the aforementioned reaction mixture of DTPA and IgG, and themixture was left standing at room temperature for 30 minutes.[In-111]DTPA-IgG and [In-111]DTPA were separated by cellulose acetateelectrophoresis, and the number of DTPA binding to IgG was calculated.The aforementioned reaction mixture of DTPA and IgG was applied to aSephadex G50 carrier to remove unbound DTPA and thereby purify DTPA-IgG.This purified DTPA-IgG was labeled with In-111 by the method describedabove, and unreacted In-111 was removed by using a Sephadex G50 carrierto obtain [In-111]DTPA-IgG. By using this [In-111]DTPA-IgG, thefollowing experiments were performed. FIG. 4 shows an autoradiographicimage of DTPA-IgG before and after purification electrophoresed bycellulose acetate electrophoresis.

2) Reaction of In-111-Labeled Monoclonal Antibody with Live HeLa Cells

5×10⁵ or 5×10⁶ of the HeLa cells were suspended in 1% BSA/PBA,[In-111]DTPA-IgG was added to the suspension, the mixture was incubatedon ice for 1 hour, the cells were washed with PBS, and radioactivitybound to the HeLa cells was measured with a gamma counter. As a result,it was found that the labeled anti-human PRDX4 monoclonal antibody didnot bind to the live HeLa cells. FIG. 5 shows specific binding ratios ofthe In-111-labeled monoclonal antibody (represented by P) bound bycontacting it with 5×10⁵ of the HeLa cells (left graph) or 5×10⁶ of theHeLa cells (right graph). IgG2a, E, and IgG1 represent negative controlsusing other antibodies. Since PRDX4 is an intracellular protein, it isreasonable that it did not bind with the HeLa cells, and it served asone basis for performing the examination using cancer-bearing animalsdescribed below.

3) Imaging in Cancer-Bearing Mouse Using In-111-Labeled MonoclonalAntibody

The HeLa cells were subcutaneously transplanted into a BALB/c-nu/numouse to form a tumor. [In-111]DTPA-IgG (50 μCi, protein amount wasadjusted to 12 μg with unlabeled IgG) was administered from the caudalvein, and X-ray images and gamma-ray images were obtained on the days 1,2, 3, and 4 after the administration. It was revealed that[In-111]DTPA-IgG accumulated in a part of the tumor with time. FIG. 6shows the X-ray images (left) and the gamma-ray images (right) of thecancer-bearing mouse obtained by anesthetizing the mouse by inhalationand fixing it with a tape. In FIG. 6, the X-ray images (left) and thegamma-ray images (right) on the days 1, 2, 3, and 4 are shown from theleft. In the X-ray images (left) of the cancer-bearing mouse on the days1 to 4 shown in FIG. 6, the sufficiently grown human cancer is imaged inthe shape of bossing near the right buttock. Further, in the gamma-rayimages (right), distinct accumulation of the labeled anti-peroxiredoxin4 (PRDX4) is observed in the tumor of the nude mouse, which isespecially distinct in the images on the day 2 to 4.

4) Confirmation of Accumulation Site of In-111-Labeled MonoclonalAntibody

After imaging on the day 4 after the administration of [In-111]DTPA-IgG,the tumor was extracted to prepare a plurality of frozen samples, andautoradiography and hematoxylin-eosin staining (HE staining) wereperformed for adjacent samples among the plurality of the preparedsamples. In FIG. 7, the autoradiographic image, the HE staining image,and an image obtained by superposing the foregoing two images are shownfrom the left. In the HE staining of the tumor section, the necroticpart around the center was stained scarcely in blue, but stained weaklyin red, and the outer normal carcinoma tissue part and thecircumferential part of the necrotic focus surrounding the center werestained in reddish blue. As shown in FIG. 7, it was revealed that[In-111]DTPA-IgG selectively accumulated in the circumferential part ofthe necrotic focus surrounding the necrotic part at the center of theextracted tumor (size: about 13.6 mm×18.3 mm). That is, from the imageobtained by superposing the autoradiographic image and the HE stainingimage, it was judged that accumulation of the In-111-labeled monoclonalantibody was observed in the circumferential part of the necrotic focussurrounding the focus of necrosis around the center.

5) Cell Morphology in In-111-Labeled Monoclonal Antibody-AccumulatingCircumferential Part of Focus of Necrosis

From the pathology of the extracted tumor, it was revealed that[In-111]DTPA-IgG accumulated in the necrotic cells in which the nucleiremained, and did not accumulate in cells of which nuclei weredeficient. It seems that this indicated that the necrosis marker proteinremained in the cells of which necrosis occurred not so much ago, andthe neighborhood thereof. That is, in FIG. 8, it can be seen that, inthe necrotic part around the center, cell morphology collapsed andnuclei were not observed; in the circumferential part of the necroticpart, although nuclei of the cells could be observed, but morphology ofthem was different from that of normal cancer cells; and in thecircumferential part around the outside of the tumor, nuclei andcytoplasm were clearly observed, and the cells were in the morphology ofnormal cancer cells. The bars in the images indicate 100 μm.

INDUSTRIAL APPLICABILITY

By using an antibody of the present invention against a necrosis marker,for example, a protein or a fragment thereof, there can be provided anovel measurement kit with which a focus of necrosis that is recognizedby the antibody or a state of progress of a pathological condition asthe cause of the necrosis can be conveniently and quickly measured. Assuch a focus of necrosis, not only foci of necrosis (tissues) of varioussolid carcinomas, myocardial infarction, and cerebral infarction, butfoci of necrosis in postnecrotic cirrhosis, necrotizing pancreatitis,necrotising fasciitis, and so forth, and further, foci of necrosis inarteriosclerotic gangrene, diabetic gangrene and obstructive gangrenecan also be an object of the kit.

As a result, development of a detection kit that can be used for a widerrange of diseases compared with various carcinoma markers etc. in viewof necrosis of lesions, or a therapeutic agent targeting necrosis partin such diseases is enabled. For example, if an artificial antibody suchas a lower molecular weight antibody is prepared, such an antibody canbe more easily incorporated into cells because it has a smallermolecular weight, whereas it is difficult to incorporate a conventionalmolecular weight antibody into cells because it has a large molecularweight. Further, such a low molecular weight antibody is considered toshow higher tissue migration properties, and therefore useful as anactive component/ingredient of a kit for diagnostic imaging or apharmaceutical composition.

1. A monoclonal antibody against a necrosis marker consisting of anexpression product of a gene encoding: the amino acid sequence of SEQ IDNO: 1, or an amino acid sequence having substitution, deletion and/orinsertion of one or several amino acid residues in the amino acidsequence of SEQ ID NO: 1 or sharing a homology of 90% or more with theamino acid sequence of SEQ ID NO: 1, and showing the same function,activity or property as that of the amino acid sequence of SEQ ID NO: 1as a protein.
 2. The monoclonal antibody according to claim 1, whereinthe necrosis marker consists of a partial polypeptide of a proteinhaving the amino acid sequence of SEQ ID NO:
 1. 3. The monoclonalantibody according to claim 1, which is produced by a hybridoma, NITEABP-1062 (YKP4 C8505 FCS(+)). 4-7. (canceled)
 8. A kit used fordetecting a focus of necrosis, which comprises the monoclonal antibodyaccording to claim
 1. 9. A conjugate for diagnostic imaging or atherapeutic treatment, which consists of a compound that specificallyreacts with a necrosis marker consisting of an expression product of agene encoding the amino acid sequence of SEQ ID NO: 1, or an amino acidsequence having substitution, deletion and/or insertion of one orseveral amino acid residues in the amino acid sequence of SEQ ID NO: 1or sharing a homology of 90% or more with the amino acid sequence of SEQID NO: 1, and showing the same function, activity or property as that ofthe amino acid sequence of SEQ ID NO: 1 as a protein, and a labelingcompound or a therapeutically effective compound.
 10. A kit for use indiagnostic imaging or a pharmaceutical composition, which comprises theconjugate according to claim 9 as an active component/ingredient. 11.The conjugate according to claim 9, wherein the compound thatspecifically reacts with the necrosis marker is a monoclonal antibodyagainst a necrosis marker consisting of an expression product of a geneencoding: the amino acid sequence of SEQ ID NO: 1, or an amino acidsequence having substitution, deletion and/or insertion of one orseveral amino acid residues in the amino acid sequence of SEQ ID NO: 1or sharing a homology of 90% or more with the amino acid sequence of SEQID NO: 1, and showing the same function, activity or property as that ofthe amino acid sequence of SEQ ID NO: 1 as a protein.